Method for producing a collagen membrane and uses thereof

ABSTRACT

The present invention relates to a method of producing a collagen membrane that has particular mechanical properties. In particular, the present invention relates to a method A of producing a collagen membrane comprising the steps of (i) isolating a collagen-containing tissue and incubating same in an ethanol solution; (ii) incubating the collagen-containing tissue from step (i) in a first solution comprising an inorganic salt and an anionic surfactant in order to denature non-collagenous proteins contained therein; (iii) incubating the collagen-containing tissue produced in step (ii) in a second solution comprising an inorganic acid until the collagen in said material is denatured; and (iv) incubating the collagen-containing tissue produced in step (iii) in a third solution comprising an inorganic acid with simultaneous mechanical stimulation for sufficient time to enable the collagen bundles in said collagen-containing tissue to align; wherein the mechanical stimulation comprises applying tension cyclically to the collagen-containing tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of PCT/AU2013/000621,filed Jun. 12, 2013, which claims benefit of Australian patentapplication 2012902458, filed Jun. 12, 2012, the entire contents ofwhich are incorporated herein by reference.

FIELD

The present invention relates to a method of producing a collagenmembrane that has particular mechanical properties. In particular, thepresent invention relates to a method of producing a collagen membranewhich comprises treating a collagen-containing tissue with inorganicsalts and anionic surfactants sufficient to produce specific mechanicalproperties.

BACKGROUND

Collagen and its derived products are used extensively in the productionof collagen-containing implantable scaffolds. Collagen is wellrecognized as a material that has low antigenicity, is biodegradable andhas good mechanical, haemostatic and cell-binding properties (Sheu etal., (2001), Biomaterials, 22(13):1713-9; Pieper et al., (2002),Biomaterials, 23(15):3183-92; Chvapil et al., (1973), Int Rev ConnectTissue Res., 6:1-61; Pachence (1996), J. Biomed. Mater. Res.;33(1):35-40; and Lee et al., (2001), Int J Pharm.; 221(1-2):1-22), whichenables it to be used to replace or repair tissue temporarily orpermanently. Collagen scaffolds are routinely used a substrate uponwhich cells are able to proliferate and differentiate and beingeventually replaced by normal tissue.

However, it is also well known that collagen-containing scaffolds canprovoke inflammation and/or fibrosis when implanted. See, for example,Wisniewski et al., (2000), J. Anal Chem.; 366 (6-7) (p. 611-621). As aconsequence, collagen-containing scaffolds are typically chemically orphysically treated (cross linked) to confer mechanical strength andresistance to enzymatic (collagenase) degradation. There are severalcross-linking strategies that have been used on collagen-containingmaterials. Glutaraldehyde is the most widely used cross-linking agent(Sheu et al., (2001) supra; Barbani et al., (1995), J Biomater. Sci.Polym. Ed.; 7(6):461-9). However, glutaraldehyde and its reactionproducts are associated with cytotoxicity in vivo, due to the presenceof cross-linking by-products and the release of glutaraldehyde-linkedcollagen peptides during enzymatic degradation (Huang-Lee et al.,(1990), J Biomed Mater Res., 24(9):1185-201; van Luyn et al., (1992),Biomaterials, 13(14):1017-24.

In order to avoid in vivo cytotoxicity of glutaraldehyde cross-linkedcollagen, several alternative compounds have been examined as potentialcollagen cross-linking agents (Khor (1997), Biomaterials, 18(2): 95-105;Sung et al. (1996), Biomaterials; 17(14): 1405-10) such as polyepoxy,hexamethylene diisocyanate (HMDI),1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC), and ultra-violet(UV) or gamma-ray irradiation. Koob et al., (2001), J Biomed Mater Res.,56(1):31-48 showed that nordihydroguaiaretic acid (NDGA) significantlyimproved the mechanical properties of synthetic collagen fibres. Inaddition, they showed that NDGA cross-linked collagen fibres did notelicit a foreign body response nor did they stimulate an immune reactionduring six weeks in vivo.

However, despite all of these advancements there remain issues withusing cross-linked collagen as well as native collagen. Thus, there isstill a need for a collagen-containing scaffold that has the followingproperties:

a) pores that interconnect in such a way as to favour tissue integrationand vascularisation;

b) biodegradability and/or bioresorbability so that normal tissueultimately replaces the scaffold;

c) surface chemistry that promotes cell attachment, proliferation anddifferentiation;

d) strength and flexibility; and

e) low antigenicity.

One area that has a particular need for a replacementcollagen-containing tissue is the repair of tympanic membrane (TM)perforations. If left untreated, TM perforations can result in hearingloss, recurrent otorrhea, possible middle ear infection and acquiredcholesteatoma (Parekh et al., (2009), The Laryngoscope; 119:1206-1213).Although most acute TM perforations heal spontaneously, large or chronicTM perforations, especially from chronic suppurative otitis media, oftenfail to heal and may require grafting (Lindeman et al., (1987), Archivesof Otolaryngology—Head and Neck Surgery; 113:1285).

Currently, surgical methods such as myringoplasty are regarded as themost effective and reliable treatment for TM perforations (Sheehy etal., (1980), The Annals of otology, rhinology, and laryngology; 89:331;Karela et al., (2008), European Archives of Oto-Rhino-Laryngology; 265:1039-1042). Various autologous grafts and allografts such as musclefascia, cartilage, perichondrium and AlloDerm have been used, however,all have their own limitations (Levin et al., (2009), Expert review ofmedical devices; 6:653-664). For instance, temporalis fascia, which isregarded as the “gold standard”, is associated with donor sitemorbidity, additional incisions, long operation time and a shortage ofmaterial in revision cases (Levin et al., (2009), supra). To date, arange of xenografts and synthetic materials, including GELFOAM® membrane(Abbenhaus, (1978), Otolaryngology; 86:ORL485), paper patch (Golz etal., (2003), Otolaryngology—Head and Neck Surgery; 128: 565) andhyaluronic acid derivatives (Teh et al., (2011), Expert Opinion onBiological Therapy; 1-14) have been investigated as suitable scaffoldsto support the regeneration of TM. However, there is little evidence tosupport any of these as optimal materials for various types ofperforations.

Moreover, several commercially available xenografts such as porcinesmall intestinal submucosa, contain xeno DNA materials and evoke aninflammatory response due to the remnant xenocellular componentsincluding serotonin. In addition, synthetic materials arenon-biodegradable, and their biomechanical and material properties aredifferent compared to the normal TM, which may affect the long-termhearing function (Levin et al., (2009), supra). Hence, there is aconstant search for better materials to achieve improved healing andhearing.

SUMMARY

The present invention provides a method of producing acollagen-containing tissue which has reduced inflammation and/orfibrosis when implanted compared to other collagen-containing tissue. Insome embodiments, the collagen-containing tissue not cross-linked.

Thus, in a first aspect the present invention provides a method ofproducing a collagen membrane comprising the steps of:

-   -   (i) isolating a collagen-containing tissue and incubating same        in an ethanol solution;    -   (ii) incubating the collagen-containing tissue from step (i) in        a first solution comprising an inorganic salt and an anionic        surfactant in order to denature non-collagenous proteins        contained therein;    -   (iii) incubating the collagen-containing tissue produced in        step (ii) in a second solution comprising an inorganic acid        until the collagen in said material is denatured; and    -   (iv) incubating the collagen-containing tissue produced in        step (iii) in a third solution comprising an inorganic acid with        simultaneous mechanical stimulation for sufficient time to        enable the collagen bundles in said collagen-containing tissue        to align;        wherein the mechanical stimulation comprises applying tension        cyclically to the collagen-containing tissue.

It will be appreciated that any inorganic salt may be used in the firstsolution as long as it is capable of forming a complex with Lewis acids.In some embodiments, the inorganic salt is selected from the groupconsisting of trimethylammonium chloride, tetramethylammonium chloride,sodium chloride, lithium chloride, perchlorate andtrifluoromethanesulfonate. In other embodiments, the inorganic salt islithium chloride (LiCl).

While any number of anionic surfactants may be used in the firstsolution, in some embodiments, the anionic surfactant is selected fromthe group consisting of alkyl sulfates, alkyl ether sulfates, alkylsulfonates, and alkyl aryl sulfonates. Particularly useful anionicsurfactants include alkyl sulphates such as sodium dodecyl sulphate(SDS).

In some embodiments, the first solution comprises about 1% (v/v) SDS andabout 0.2% (v/v) LiCl.

In some embodiments, the inorganic acid in the second solution comprisesabout 0.5% (v/v) HCl, while the inorganic acid in the third solutioncomprises about 1% (v/v) HCl.

It will be appreciated by those skilled in the art that the incubationperiods in each of the three steps will vary depending upon: (i) thetype of collagen-containing tissue; (ii) the type of inorganic salt/acidand/or anionic surfactant; (iii) the strength (concentration) of eachinorganic salt/acid and/or anionic surfactant used and (iv) thetemperature of incubation. In some embodiments, the incubation period instep (i) is at least 8 hours. In other embodiments, the incubationperiod in step (ii) is less than 60 minutes, while in other embodimentsthe incubation period in step (iii) is at least 20 hours.

In some embodiments, the incubation in step (ii) is at about 4° C. Inother embodiments, the incubation in step (ii) is undertaken for atleast 12 hours.

In some embodiments, the second solution comprises about 0.5% (v/v) HCl.

In some embodiments, the incubation in step (iii) is undertaken forabout 30 minutes. In other embodiments, the incubation in step (iii) isundertaken with shaking.

In some embodiments, the third solution comprises about 1% (v/v) HClsolution.

In some embodiments, the incubation in step (iv) is undertaken for about12 to 36 hours, preferably for about 24 hours. In other embodiments, theincubation in step (iv) is undertaken with shaking.

In some embodiments, the methods of the invention further comprises aneutralization step between step (iii) and step (iv) which comprisesincubation of said collagen-containing tissue with about 0.5% (v/v)NaOH.

In some embodiments, the methods of the invention further comprises step(v) which comprises incubating the collagen-containing tissue from step(iv) with acetone and then drying the collagen-containing tissue.

In some embodiments, the methods of the invention further comprisesbetween steps (ii) and (iii) and/or between steps (iii) and (iv) a stepof contacting the collagen-containing tissue with glycerol in order tovisualise and facilitate the removal of fat and/or blood vessels.

The glycerol may be contacted with the collagen-containing tissue forany amount of time that will facilitate the removal of fat and/or bloodvessels. In some embodiments, the contact time is at least 10 minutes.

In some embodiments, the methods of the invention further comprisesbetween steps (ii) and (iii) and/or between steps (iii) and (iv) a washstep for the collagen-containing tissue. The purpose of the wash stepused between steps (ii) and (iii) is to remove denatured proteins. Thus,any wash solution capable of removing denatured proteins can be used. Insome embodiments the wash solution used between steps (ii) and (iii) isacetone.

Following the washing with acetone, the collagen-containing tissue isfurther washed with sterile water.

In some embodiments, the collagen-containing tissue is further washed ina NaOH:NaCl solution. If the collagen-containing tissue is washed withNaOH:NaCl it is then preferably washed with sterile water.

In some embodiments, after step (iv) the collagen-containing tissue isfurther washed with the first solution.

It will be appreciated by those skilled in the art that thecollagen-containing tissue can be any tissue isolated from a mammaliananimal. However, it will also be appreciated that thecollagen-containing tissue will comprise dense connective tissue. Insome embodiments, the collagen-containing tissue is isolated from asheep, a cow, a pig or a human. Preferably, the collagen-containingtissue is isolated from a human.

In some embodiments, the collagen-containing tissue is autologous.

In a second aspect, the present invention provides a collagen membraneproduced by a method to the first aspect, wherein said membrane producedby the method comprises greater than 80% (w/w) type I collagen fibres orbundles having a knitted structure and a modulus of greater than 300MPa.

In some embodiments, the collagen membrane will have a modulus ofgreater than 400 MPa and preferably greater than 500 MPa.

The collagen membrane will also have an extension at maximum load ofless than 85%, preferably less than 80%.

In a third aspect, the present invention provides a method for preparinga device for implantation into the body or tissue of a person or animal,said method comprising placing a collagen membrane on said device,wherein said collagen membrane is produced by a method comprising:

-   -   (i) isolating a collagen-containing tissue and incubating same        in an ethanol solution;    -   (ii) incubating the collagen-containing tissue from step (i) in        a first solution comprising an inorganic salt and an anionic        surfactant in order to denature non-collagenous proteins        contained therein;    -   (iii) incubating the collagen-containing tissue produced in        step (ii) in a second solution comprising an inorganic acid        until the collagen in said material is denatured; and    -   (iv) incubating the collagen-containing tissue produced in        step (iii) in a third solution comprising an inorganic acid with        simultaneous mechanical stimulation for sufficient time to        enable the collagen bundles in said collagen-containing tissue        to align;        wherein the mechanical stimulation comprises applying tension        cyclically to the collagen-containing tissue.

In a fourth aspect the present invention provides a device havingenhanced biocompatibility for implantation into the body or tissue of aperson or animal, wherein said device comprises a collagen membrane isproduced by a method comprising:

-   -   (i) isolating a collagen-containing tissue and incubating same        in an ethanol solution;    -   (ii) incubating the collagen-containing tissue from step (i) in        a first solution comprising an inorganic salt and an anionic        surfactant in order to denature non-collagenous proteins        contained therein;    -   (iii) incubating the collagen-containing tissue produced in        step (ii) in a second solution comprising an inorganic acid        until the collagen in said material is denatured; and    -   (iv) incubating the collagen-containing tissue produced in        step (iii) in a third solution comprising an inorganic acid with        simultaneous mechanical stimulation for sufficient time to        enable the collagen bundles in said collagen-containing tissue        to align;        wherein the mechanical stimulation comprises applying tension        cyclically to the collagen-containing tissue.

Once produced, the collagen membrane produced by the methods of thepresent invention can be used to repair various tissue defects.

Accordingly, in a fifth aspect the present invention provides use of acollagen membrane according to the first or second aspect or a deviceaccording to the fourth aspect for the repair of a tissue defect in amammalian animal.

In a sixth aspect, the present invention provides a method of treating atissue defect in a mammalian animal subject comprising the step ofinserting a collagen membrane according to the first or second aspect ora device according to the fourth aspect into said tissue defect.

The methods of the present invention can be used to produce collagenmembranes of various thicknesses depending upon their end use. Forexample, membranes for use in the repair of tympanic membranes innon-human animals might be 50 μm thick, while repair of tympanicmembranes in humans might be 100 μm thick. Thus, various membranethicknesses are envisaged.

In a seventh aspect, the present invention provides a collagen membraneproduced by the method of the first aspect that is at least 10 μm.Preferably, the membrane is between about 10 μm and 400 μm thick. Morepreferably, between 50 μm and 200 μm thick. In some embodiments, thecollagen membrane of the present invention is about 100 μm thick.

In an eighth aspect the present invention provides a method of repairinga tympanic membrane perforation comprising the step of inserting acollagen membrane according to the first or second aspect or a deviceaccording to the fourth aspect into or adjacent to said tympanicmembrane perforation.

In some embodiments, the method of the first or second aspects has theproviso that no cross-linking of the collagen-containing tissue takesplace. In some embodiments, the method of the first or second aspectshas the proviso that no glutaraldehyde is used in the methods of thepresent invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the surface morphology of the collagen membrane produced bythe methods of the present invention (TYMPACOL™ membrane referred to asACS herein) compared to other membranes. Scanning electron microscopyshows the surface morphology of three membranes (Panel A-C; ×500, D;×200). TYMPACOL™ membrane (referred to as ACS in FIG. 1) possesses twodistinct surfaces, a smooth surface featuring compact collagen bundles(Panel A), and a rough, porous surface of loose collagen fibres (PanelB). Paper patch (membrane) surface is uneven with few small pores (PanelC). GELFOAM® membrane shows substantial pores of varying sizes (PanelD). Scale bar: 500 μm.

FIG. 2 shows scanning electron microscopy (SEM) image (×100) of acollagen membrane produced by the methods of the present invention.

FIG. 3 shows scanning electron microscopy (SEM) image (.times.200) of acommercially available bioscaffold (“BIO-GIDE™” membrane) LuitpoldPharmaceuticals, Inc, Shirley, N.Y. USA.

FIG. 4 shows a bar graph showing comparative mean maximum load for acollagen membrane produced by the methods of the present invention andcommercially BIO-GIDE™ membrane.

FIG. 5 shows a bar graph showing comparative mean extension at maximumload for a collagen membrane produced by the methods of the presentinvention and commercially BIO-GIDE™ membrane.

FIG. 6 shows a bar graph showing comparative mean load at yield for acollagen membrane produced by the methods of the present invention andcommercially BIO-GIDE™ membrane.

FIG. 7 shows a bar graph showing comparative mean extension at yield fora collagen membrane produced by the methods of the present invention andcommercially BIO-GIDE™ membrane.

FIG. 8 shows photomicrographs of healed tympanic membranes (TMs) 28 daysfollowing grafting of a collagen membrane produced by the methods of thepresent invention compared to other commercially available membranes.Normal TMs stained with H&E (Panel A) and Masson trichrome (MT) (PanelC) are shown. At 28 days, TMs treated with TYMPACOL™ membrane (ACS(Panels B & D)) had normal trilaminar structure, consisting of dense andwell-organized collagen bundles in the CT layer. TMs treated with paperpatch (Panels E, H) and GELFOAM® membrane (Pfizer, Puurs, Belgium)(Panels F, I) remained thickened in the healed area with loose anddisorganized collagen fibres in the middle layer. TMs in the controlgroup (Panels G, J) remained thick with atypical structure and regionsof irregular collagen fibres. At 14 days, all TMs were significantlythickened compared to the normal TM (Panel K). By 28 days, TMs thicknessin the ACS groups showed no significant differences compared to thenormal TMs (Panel L). (*p<0.05, **p<0.01). Arrowheads indicate theresidual scaffolds. H&E and Masson trichrome staining Scale bars: 50 μm.

FIG. 9 shows auditory brainstem responses assessment of hearing recoveryfollowing grafting. The hearing recovery was defined as the differencebetween auditory threshold immediately following perforation(pre-repair) and at specific time points following grafting(post-repair). The values represent mean±standard error of mean (SEM)(n=5). Hearing recovery following grafting in each group was performedusing multiple linear regression analysis. Auditory threshold of allrats recovered over time and significant differences were observed whencomparing between different treatments (p<0.01). Hearing in the ratstreated by the ACS recovered significantly faster compared to thosetreated with paper patch, GELFOAM® membrane and spontaneous healing(control). Statistical significance between groups was: a ACS andspontaneous healing (p<0.01); ACS and paper (p<0.01); ACS and GELFOAM®membrane (p<0.01).

FIG. 10 shows healing of tympanic membrane perforation at different timepoints following grafting.

Further features, advantages and details of the present invention willbe appreciated by those of ordinary skill in the art from a reading ofthe figures and the detailed description of the embodiments that follow,such description being merely illustrative of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Generally stated, embodiments of the subject invention are directed tocollagen membrane, coverings, coatings and/or scaffolds which areparticularly suitable for implantable medical devices, and methods ofmaking and using the same in animal or human patients. The patient canbe a human or other animal, such as a primate, equine, bovine, ovine,canine, or feline animal. The collagen membrane, coatings, coveringsand/or scaffolds can be provided as a tissue-contacting surface whichmay encapsulate all or a portion of the implantable devices to therebyprovide a reduced immunogenic response and/or long-lived in vivofunctionality of the implanted device.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items. As used herein, phrases such as “between X and Y” and“between about X and Y” should be interpreted to include X and Y. Asused herein, phrases such as “between about X and Y” mean “between aboutX and about Y”. As used herein, phrases such as “from about X to Y” mean“from about X to about Y”.

The term “about” as used herein refers to a deviation in the valuefollowing the term by 10% above or below. For example, reference toabout 70% ethanol includes ranges between 63% and 77% i.e. 10% below orabove the 70% value. This includes 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76% and 77% ethanol.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the specification andrelevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. Well-known functions orconstructions may not be described in detail for brevity and/or clarity.

It will be understood that when an element is referred to as being “on”,“attached” to, “connected” to, “coupled” with, “contacting”, etc.,another element, it can be directly on, attached to, connected to,coupled with or contacting the other element or intervening elements mayalso be present. In contrast, when an element is referred to as being,for example, “directly on”, “directly attached” to, “directly connected”to, “directly coupled” with or “directly contacting” another element,there are no intervening elements present. It will also be appreciatedby those of skill in the art that references to a structure or featurethat is disposed “adjacent” another feature may have portions thatoverlap or underlie the adjacent feature.

It will be understood that, although the terms first, second, etc. maybe used herein to describe various elements, components, regions, layersand/or sections, these elements, components, regions, layers and/orsections should not be limited by these terms. These terms are only usedto distinguish one element, component, region, layer or section fromanother region, layer or section. Thus, a first element, component,region, layer or section discussed below could be termed a secondelement, component, region, layer or section without departing from theteachings of the present invention. The sequence of operations (orsteps) is not limited to the order presented in the claims or figuresunless specifically indicated otherwise.

The term “implantable” means the “collagen-containing tissue”, “collagenmembrane”, “device” or “scaffold” can be inserted, embedded, grafted orotherwise acutely or chronically attached or placed on or in a patient.The term “collagen-containing tissue” means skin, muscle and the likewhich can be isolated from a mammalian body that contains collagen. Theterm “collagen-containing tissue” also encompasses “synthetically”produced tissue in which collagen or collagen containing material hasbeen assembled or manufactured outside a body.

The term “collagen membrane” is intended in this connection to beunderstood to mean a membrane chiefly based on collagen. A “membrane”typically comprises the components as described herein.

The term “chronically” means that the “collagen-containing tissue”,“collagen membrane”, “device” or “scaffold” is configured to remainimplanted for at least 2 months, typically at least 6 months, and insome embodiments, one or more years while remaining operational for itsintended function. The terms “coating” or “covering” refer to a materialon a target surface of the membrane, device or scaffold. The coating canbe a porous coating that can inhibit cell and tissue fouling of theunderlying membrane, device or scaffold. The coating may not promotetissue growth. The coating can be a thin or thick film, foam or otherbarrier to tissue fouling and biodegradation. The term “scaffold” refersto a porous material and/or structure into which cells, tissue, vessels,etc, can grow into, colonize and populate.

Collagen bundles are composed of collagen fibres. Collagen fibres arecomposed of three polypeptide chains that intertwine to form aright-handed triple helix. Each collagen polypeptide chain is designatedas an α chain and is rich in glycine, proline and hydroxyproline. Thereare a number of different α chains and different combinations of these αchains correspond with different types of collagen. In some embodiments,the collagen membrane of the present invention comprises type Icollagen. Type I collagen is composed of two α1 chains and one α2 chain.

In some embodiments, the collagen fibres or bundles are provided fromdense connective tissue isolated from a source. The term “denseconnective tissue” as used herein refers to the matrix comprisedprimarily of type I collagen fibres or bundles found in the tendons,ligaments and dermis of all mammals. Dense connective tissue is distinctfrom “loose connective tissue”. Loose connective tissue is characterisedby loosely arranged fibres and an abundance of cells and is present, forexample, beneath the epithelia that covers body surfaces and linesinternal organs.

Dense connective tissue may be regular or irregular. Dense regularconnective tissue provides strong connection between different tissuesand is found in tendons and ligaments. The collagen fibres in denseregular connective tissue are bundled in a parallel fashion. Denseirregular connective tissue has fibres that are not arranged in parallelbundles as in dense regular connective tissue and comprises a largeportion of the dermal layer of skin. The collagen membrane of thepresent invention may be composed of either regular dense connectivetissue or dense irregular connective tissue, or a combination of both.

Collagen “microfibrils,” “fibrils,” “fibres,” and “natural fibres” referto naturally-occurring structures found in a tendon. Microfibrils areabout 3.5 to 50 nm in diameter. Fibrils are about 50 nm to 50 μm indiameter. Natural fibres are above 50 μm in diameter. A “syntheticfibre” refers to any fibre-like material that has been formed and/orchemically or physically created or altered from its naturally-occurringstate. For example, an extruded fibre of fibrils formed from a digestedtendon is a synthetic fibre but a tendon fibre newly harvested from amammal is a natural fibre. Of course, synthetic collagen fibres caninclude non-collagenous components, such as hydroxyapatite or drugs thatfacilitate tissue growth. For example, the compositions can containgrowth factors such as basic fibroblast growth factor, tumour growthfactor beta, bone morphogenic proteins, platelet-derived growth factor,and insulin-like growth factors; chemotactic factors such fibronectinand hyaluronan; and extracellular matrix molecules such as aggrecan,biglycan, and decorin. Of course, synthetic collagen fibres can includenon-collagenous components, such as particulates, hydroxyapatite andother mineral phases, or drugs that facilitate tissue growth. Forexample, the compositions can contain carbon nano-tubes, zincnano-wires, nano-crystalline diamond, or other nano-scale particulates;larger crystalline and non-crystalline particulates such as calciumphosphate, calcium sulfate, apatite minerals. For example, thecompositions can contain therapeutic agents such as bisphosphonates,anti-inflammatory steroids, growth factors such as basic fibroblastgrowth factor, tumour growth factor beta, bone morphogenic proteins,platelet-derived growth factor, and insulin-like growth factors;chemotactic factors such fibronectin and hyaluronan; and extracellularmatrix molecules such as aggrecan, biglycan, and decorin.

The term “source” as used herein refers to any collagen tissuecontaining dense connective tissue in any mammal. In some embodiments,the tissue containing dense connective tissue is a tendon. A tendon isthe tissue which connects muscle to bone in a mammal.

In some embodiments, the collagen-containing tissue may be isolated fromany mammalian animal including, but not limited to a sheep, a cow, a pigor a human. In other embodiments, the collagen-containing tissue isisolated from a human.

In some embodiments, the collagen-containing tissue is “autologous”,i.e. isolated from the body of the subject in need of treatment.

In some embodiments, the present invention provides a collagen membranecomprising greater than 80% type I collagen. In other embodiments, thecollagen membrane comprises at least 85% type I collagen. In still otherembodiments the collagen membrane comprises greater than 90% type Icollagen.

The collagen fibres or bundles of the collagen membrane form a knittedstructure. The term “knitted structure” as used herein refers to astructure comprising first and second groups of fibres or bundles wherefibres or bundles in the first group extend predominately in a firstdirection and fibres or bundles in the second group extend predominatelyin a second direction, where the first and second directions aredifferent to each other and the fibres or bundles in the first groupinterleave or otherwise weave with the fibres or bundles in the secondgroup. The difference in direction may be about 90°.

The term “maximum tensile load strength” as used herein refers to themaximum tensile load that the collagen membrane can bear. On a Load vExtension curve this is represented by the peak load on the curve.

In some embodiments, the collagen membrane has maximum tensile loadstrength of greater than 20 N. In some embodiments, the collagenmembrane of the present invention has maximum tensile load strengthgreater than 25 N, 40 N, 60 N, 80 N, 100 N, 120 N or 140 N.

Further, it is believed that the knitted structure of the embodiments ofthe collagen membrane provides reduced extension at maximum load of thebioscaffold while providing an increase in modulus.

The term “modulus” as used herein means Young's Modulus and isdetermined as the ratio between stress and strain. This provides ameasure of the stiffness of the collagen membrane.

In some embodiments the collagen membrane has a modulus of greater than100 MPa. In other embodiments the collagen membrane has a modulus ofgreater than 200 MPa, 300 MPa, 400 MPa, or 500 MPa.

The term “extension at maximum load” as used herein means the extensionof the collagen membrane at the maximum tensile load strength referencedto the original length of the collagen membrane in a non-loadedcondition. This is to be contrast with maximum extension which will begreater.

In some embodiments, the collagen membrane has extension at maximum loadof less than 85% of the original length.

Examples of devices that can benefit from the collagen membrane,collagen coatings and/or scaffolds contemplated by embodiments of theinvention, include, but are not limited to, implantable stents,including cardiac, arterial, neuro (brain), urinary, and other stents,implantable power generators (IPGs), pacemakers, defibrillators,cardioverters, stimulators and/or lead systems for the brain, centralnervous system (CNS) or peripheral nervous system, cardiac or otherbiological system, cardiac replacement valves, implantable sensorsincluding glucose sensors, cardiac sensors, identity or tracking sensors(e.g., RFID), sensors to detect or measure O₂, pH, temperature, ions,and the like, orthopaedic implants, including tissue implants, such asfacial implants for the chin, cheek, jawbone, and nose, implantablesubcutaneous or percutaneous access ports, drain tubes such asEustachian drain tubes, catheters such as urinary catheters,respiratory-assist tubes, and the like.

The collagen membrane, scaffold or covering of fibres can be configuredto substantially encase the target implantable device or may cover onlya portion thereof.

The collagen membrane, scaffold or covering can be a three dimensionalarray of fibres or fibrils held together or on the device in anysuitable manner including by their natural affinity to stick togetherupon compression or extrusion, by using a sticky coating or adhesive,such as a gelatinous coating, or by otherwise attaching the fibres toform the array.

The term “simultaneous mechanical stimulation” used in the methodsdescribed herein refers to the process of stretching the collagenmembrane during the chemical processing of the collagen-containingtissue. The membrane may undergo static and/or cyclic stretching.Accordingly, in some embodiments the simultaneous mechanical stimulationmay comprise:

(i) stretching of the membrane for a preset period;

(ii) relaxation of the membrane for a preset period; and

(iii) n-fold repetition of steps (i) and (ii), where n is an integergreater than or equal to 1.

If the mechanical stimulation is carried out by stretching the membrane,the membrane is preferably stretched along its long axis.

In some embodiments, the simultaneous mechanical stimulation comprisesapplying tension cyclically to collagen-containing tissue, wherein theperiodicity of the tension comprises a stretching period of about 10seconds to about 20 seconds and a relaxing period of about 10 seconds,and the strain resulting therefrom is approximately 10%, and themechanical stimulation continues until the collagen bundles within thecollagen-containing tissue are aligned as described herein.

The subject invention also concerns the use of collagen membranes orscaffolds of the invention for the in vitro or in vivo delivery ofbioactive compounds, drugs, growth factors, proteins, peptides, nucleicacids, inorganic or organic molecules, etc. A collagen-containing tissueor scaffold of the invention can be loaded with a bioactive compound,etc. and then the loaded scaffold can be implanted or contacted with thebody, tissue, cells, etc. of a person or animal. The compounds are thenpermitted to be released from the scaffold into the body, tissue, cell,etc. The collagen membrane or scaffold can be provided on abiodegradable or non-degradable support structure or matrix.

The collagen used in the present invention can be synthetic or derivedfrom any suitable animal species. The collagen can be from a vertebrateanimal or an invertebrate (e.g., starfish, sea urchin, sponges, etc.).In some embodiments, the collagen is fish, shark, skate, or raycollagen. In another embodiment, the collagen is human, equine, bovine,ovine, porcine, canine, or feline collagen. In an exemplifiedembodiment, the collagen is bovine collagen.

Collagen-containing tissue or scaffolds of the present invention arestable both in vitro and in vivo for at least 4 weeks at bodytemperature.

The terms “repairing” or “repair” or grammatical equivalents thereof areused herein to cover the repair of a tissue defect in a mammaliananimal, preferably a human. “Repair” refers to the formation of newtissue sufficient to at least partially fill a void or structuraldiscontinuity at a tissue defect site. Repair does not however, mean orotherwise necessitate, a process of complete healing or a treatment,which is 100% effective at restoring a tissue defect to its pre-defectphysiological/structural/mechanical state.

The term “tissue defect” or “tissue defect site”, refers to a disruptionof epithelium, connective or muscle tissue. A tissue defect results in atissue performing at a suboptimal level or being in a suboptimalcondition. For example, a tissue defect may be a partial thickness orfull thickness tear in a tendon or the result of local cell death due toan infarct in heart muscle. A tissue defect can assume the configurationof a “void”, which is understood to mean a three-dimensional defect suchas, for example, a gap, cavity, hole or other substantial disruption inthe structural integrity of the epithelium, connective or muscle tissue.In certain embodiments, the tissue defect is such that it is incapableof endogenous or spontaneous repair. A tissue defect can be the resultof accident, disease, and/or surgical manipulation. For example,cartilage defects may be the result of trauma to a joint such as adisplacement of torn meniscus tissue into the joint. Tissue defects maybe also be the result of degenerative diseases such as osteoarthritis.

Typically, the collagen membrane of the invention will be implanted atthe site of the tissue defect and secured in place by any conventionalmeans known to those skilled in the art, e.g. suturing, suture anchors,bone fixation devices and bone or biodegradable polymer screws.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1 Method for the Manufacture of Collagen Membrane

A collagen segment from porcine inner organ lining was carefullyseparate and placed into a solution comprising about 70% ethanol andallowed to briefly incubate at room temperature. The collagen-containingtissue was then stretched fatty side up over the working surface and asmuch fat tissue and blood vessels as possible was removed.

In order to visualize fat tissue present the collagen-containing tissuewas coated with glycerol for about 10 minutes. At which point thecollagen was transparent, but the fat tissue was a white colour. Usingforceps we separated the white fat tissue from the collagen under ananatomical microscope.

When complete, the collagen-containing tissue was carefully transferredto a sealed container and incubated in a solution comprising about 1%(v/v) SDS and 0.2% (v/v) LiCl in order to denature the non-collagenousproteins. The incubation was left overnight at 4° C.

The collagen-containing tissue was then carefully washed two times in100% acetone to remove the denatured the non-collagenous proteins. Thetissue was then centrifuged at 100 RPM in a 200 ml container in order togently spin down residual solutions, non-collagenous proteins andnucleic acids from the collagen-containing tissue.

The collagen-containing tissue was carefully removed and once againwashed in membranes Steripure™ water 3 times.

Sometimes, we also washed the collagen-containing tissue in a solutioncomprising NaOH:NaCl after which we centrifuged the tissue at 100 RPMfor 90 minutes.

The collagen-containing tissue was then immersed in 0.5% (v/v) HCl andplaced on shaker for 30 minutes to denature the collagen. We found thatthe concentration of HCl and incubation time was important in order toavoid damaging the mechanical structure of the resulting tissue.

The collagen-containing tissue was then removed and once again washed inSteripure™ water 3 times.

The collagen-containing tissue was then neutralized using 0.5% (v/v)NaOH. At this stage preliminary testing of the mechanical properties ofresulting collagen-containing tissue could be undertaken.

The collagen-containing tissue was then manipulated using mechanicalforces (compression and extension) using a stainless steel frame. Oncethe collagen-containing tissue was stretched to the right size,thickness and the like, the tissue was denatured in situ i.e. within theframe, immersion in a solution comprising 1% (v/v) HCl. Typically, thetissue was incubated with shaking at 100 RPM for 22-25 hours until thecollagen fibre bundles had aligned.

The collagen-containing tissue was then washed with water and rinsedwith mixture of 1% (v/v) SDS and 0.2% (v/v) LiCl.

Depending upon the end use, the collagen-containing tissue was thenre-coated with glycerol for 10 minutes to visualise any residual fattissue. As above, forceps were used to separate the remaining white fattissue from the collagen under an anatomical microscope. Any extracollagen bundles are also removed at this stage in order to control thethickness of the collagen-containing tissue.

Finally, the collagen-containing tissue was treated with acetone andair-dried while still stretched within the frame so that the alignedcollagen bundles became fixed. The collagen-containing tissue was thenstretched, compressed and/or rolled to create a smooth surface. Thefinished collagen membrane tissue was then examined and cut to sizeusing a laser cutter.

SEM was performed to characterize the surface morphology of the collagenmembrane compared to other types of membranes. In brief, the tissuesamples were sputter-coated with 5 nm thick platinum (SEM coating unit,E 1020, Hitachi Science Systems Ltd., Japan) and both sides were viewedunder a scanning electron microscope (S260, Leica, Cambridge, England)at a low voltage (20 kV).

FIG. 1 shows the surface morphology of the collagen membrane produced bythe methods of the present invention (TYMPACOL™ membrane referred to asACS herein) compared to other membranes. Scanning electron microscopyshows the surface morphology of three scaffolds (Panel A-C; .times.500,D; .times.200). TYMPACOL™ membrane (referred to as ACS in FIG. 1)possesses two distinct surfaces, a smooth surface featuring compactcollagen bundles (Panel A), and a rough, porous surface of loosecollagen fibres (Panel B). Paper patch (membrane) surface is uneven withfew small pores (Panel C). GELFOAM® membrane shows substantial pores ofvarying sizes (Panel D). Scale bar: 500 μm.

FIG. 2 shows scanning electron microscopy (SEM) image (×100) of acollagen membrane produced by the above method.

FIG. 3 shows scanning electron microscopy (SEM) image (.times.200) of acommercially available bioscaffold (“BIO-GIDE™” membrane) LuitpoldPharmaceuticals, Inc, Shirley, N.Y. USA. It can be seen that thecollagen bundle arrangement in the BIO-GIDE™ membrane is less uniformedthan the TYMPACOL™ membrane.

FIG. 4 shows a bar graph showing comparative mean maximum load for acollagen membrane produced by the methods of the present invention andcommercially BIO-GIDE™ membrane.

FIG. 5 shows a bar graph showing comparative mean extension at maximumload for a collagen membrane produced by the methods of the presentinvention and commercially BIO-GIDE™ membrane.

FIG. 6 shows a bar graph showing comparative mean load at yield for acollagen membrane produced by the methods of the present invention andcommercially BIO-GIDE™ membrane.

FIG. 7 shows a bar graph showing comparative mean extension at yield fora collagen membrane produced by the methods of the present invention andcommercially BIO-GIDE™ membrane.

Conclusions

We found that the above method has benefits over traditionalalkaline-acid methods of treating collagen-containing tissue as follows:

1) The incubation with a solution comprising 1% SDS and 0.2% LiCl enablethe denaturation and removal of non-collagenous proteins and nucleicacids which are known to cause an inflammatory response with otherimplantable membranes.

2) Glycine coating enable the separation of fat tissue from thecollagen-containing tissue which, if not removed, causes issues with theflexibility of the tissue.

3) The use of HCl together with incubation time enabled us to produce amembrane with the appropriate mechanical properties without the use ofcross-linking agents such as glutaraldehyde.

4) The mechanical forces applied to the collagen membrane while it wasfixed to the frame enabled us to re-arrange the collagen bundles andfibres necessary for the formation of the special structure.

5) The macro and microscopic examination on the direction of collagenbundles in the and the collagen-containing tissue shows a collagenbundle structural orientation that makes the tissue more useful inimplantation studies.

Example 2 Characterisation of Collagen Membrane Compared to OtherMembrane

A 40 μm thick sample of TYMPACOL™ membrane produced by the method inExample 1, was used in a clinical trial compared to commerciallyavailable membranes. The commercial products included:

1). Paper patch, which was obtained from cigarette paper (Tally Ho,Imperial Tobacco Australia, Australia) and is approximately 20 μm thick,white and opaque;

2). GELFOAM® membrane (absorbable gelatine sponge, Pharmacia & UpjohnInc, New York, USA), is a highly absorbent, non-elastic sponge which isaround 4 mm thick with pore size varying between 30-700 μm (Rohanizadehet al., (2008), J. Materials Science; 19: 1173-1182.

Male Sprague-Dawley rats, weighing 250-300 grams, were used for theclinical trial according to the institutional animal ethic approval.Prior to the study, all animals were inspected using a S5 modelotomicroscope (Zeiss, Germany) to ensure they were free of middle earpathology. Animals were randomly divided into four scaffold repairgroups, namely TYMPACOL™ membrane (n=30), paper patch (n=30), GELFOAM®membrane (n=30) and control (spontaneous healing) (n=30). In addition, agroup of ten rats (n=10) were allocated as normal controls (without anyperforation or scaffold).

All the surgical procedures were performed under general anaesthesiawith intramuscular Ketamine (80 mg/kg) and Medetomidine (0.5 mg/kg).Debris from the external auditory canal was removed using a 3.0 mm auralspeculum and the external auditory canals were prepped with povidoneiodine solution. Bilateral tympanic membrane (TM) perforations,measuring approximately 1.8 mm in diameter, were created using a sterile23-gauge needle in the posterior half of the pars tensa via a transcanalapproach. Four different materials were then trimmed into pieces (2.4 mmin diameter), rinsed with 1× phosphate buffered saline solution (pH 7.4)(Invitrogen, Shanghai, China), and grafted onto the right TM perforationusing on-lay myringoplasty. The left ear served as an internal controlwhere no graft material was placed on the perforated TM. All rats weregiven subcutaneous buprenorphine (0.02-0.08 mg/kg) for postoperativeanalgesia.

The TM healing of different treatment groups was evaluated by otoscopy,scanning electron microscopy (SEM), histology and transmission electronmicroscopy (TEM), while the hearing function was analysed by auditorybrainstem responses (ABR). In each group, the same five rats (n=5) wereselected randomly for both otoscopic and ABR assessment at 3, 5, 7, 9,14, and 28 days postoperatively. In these subgroups of five rats, threewere used for histological evaluation, and one each for SEM and TEM.

Otoscopic Observation

To investigate TM healing an acute rat model of TM perforation wasestablished. Five rats from each group were randomly chosen at each timepoint for otoscopic observation using a digital video otoscope (MedRX,Largo, Fla.) under general anaesthesia. The TMs were viewed by twoindependent observers with respect to perforation closure, infection,myringosclerosis, granulation tissue and thickening. Each TM perforationwas graded as either completely closed or unclosed. Only TMs that hadcompletely closed were considered healed. Digital images were recordedusing Aurisview software (Ear Science Institute Australia, Subiaco,Australia).

SEM was performed to evaluate the healing process of TM following repairby scaffolds. Briefly, the rat TM specimens were fixed with 2.5%glutaraldehyde in 4° C. overnight, dehydrated in ethanol solutionsfollowed by critical point drying (HCP-2, Hitachi, Tokyo, Japan).Finally, the samples were coated with 5 nm thick platinum where themedial surface of the TMs was observed under SEM.

All rats survived the surgical procedures with no complicationspostoperatively. The lateral aspect of TMs was observed via an otoscopeto assess the effect of grafting at each time point. No signs ofinfection or abnormalities were observed in any of the rats.

In the control group, the TMs appeared thicker and opaquepost-perforation, with prominent microvessels visible close to theperforation margin. By 14 days, the TM became increasingly transparentand majority of the perforations had fully closed. At 28 days, all theperforations were completely healed, although visible scars resemblingan opalescent ring were observed at the perforation site. Thesemi-transparency of TYMPACOL™ membrane allowed direct observation ofthe TM healing. Throughout the healing process, TYMPACOL™ membraneretained its structural stability and adhered well to the TM remnant.The opacification of TM and microvessel was less pronounced compared tothose in the control group. The perforations had healed as early as 7days after grafting where the healed TMs appeared normal. In contrast,paper patch and GELFOAM® membrane were opaque, making it difficult toexamine the middle ear during healing. Moreover, these materials tendedto detach easily from the healing TM. In particular, the bulk ofGELFOAM® membrane shrank and its porous structure was lost over time. At28 days, the TMs in the paper patch and GELFOAM® membrane groupsappeared healed but with some scarring.

Following sacrifice at individual time points, closure of theperforation was confirmed by observing the internal surface of theharvested TMs using an otomicroscope. TM healing in the TYMPACOL™membrane groups was markedly quicker compared to the other groups (FIG.10). 60% (3/5) of TYMPACOL™ membrane treated ears were completely healedbut none in the control group had healed (0/5) (p<0.05). After 9 days,the TM was completely healed in all five rats in the TYMPACOL™ membraneand paper patch groups, which was significantly different compared tothe control group (2/5) (p<0.05). At 14 days, all ears were completelyhealed except one TM in the control group (4/5). By 28 days postsurgery, all the TMs had completely healed.

Histological Evaluation

Following sacrifice, both external ears were separated at theosteocartilaginous junctions and the TMs along with the bony annuluswere removed from the tympanic bulla. Harvested specimens were fixed in10% neutral buffered formalin for 24 hours followed by decalcificationin 10% ethylenediaminetetraacetic acid solution (EDTA) (pH 7.4) for twoto three weeks. Decalcified TMs were dehydrated in a series of gradedalcohols, embedded in paraffin wax and transversely sectioned at athickness of 4 μm. All sections were evaluated using haematoxylin andeosin (H&E) staining. Masson's trichrome staining was performed toexamine the morphology of collagen fibres. All stained slides weredigitally scanned using an Aperio ScanScope XT automated slide scanner(Aperio Technologies Inc., Vista, Calif.; 40×/0.75 Plan Apo objective).Images were saved as TIFFs for histological evaluation. TM thickness ofhealed TM sections of day 14 and 28 was measured using Aperio ImageScopeViewer software.

The histology of the TM healing and effects of the four scaffolds wereexamined over 28 days. Compared to other groups, TM healing in thecontrol group was relatively slower. In the first week, the perforationremained patent, although hyperplasia was observed in the epithelial andconnective tissue (CT) layers of the TM. On day 5, a keratin spur wasseen and the perforations started to close at 9 days with significantthickening throughout the three TM layers. By 28 days, the healed TMbecame thinner but with residual thickening at the previous perforationsite. The CT layer was found to be disorganized with loosely packedcollagen fibres (FIG. 8).

In the TYMPACOL™ membrane treated group, epithelial hyperplasia andvascular proliferation were evident in the early stages. Infiltratingcells resembling fibroblasts were abundant in the CT layer withoccasional lymphocytes surrounding the graft. At 28 days, the healed TMappeared normal with a trilaminar structure (FIG. 8).

In contrast, numerous inflammatory cells (predominantly lymphocytes) andprominent exudate was observed surrounding the paper patch. Although theTM perforation eventually healed, the TM remained thickened withdisorganization of the newly synthesized fibres (FIG. 8). Likewise,GELFOAM® membrane induced the infiltration of inflammatory cells atimplanted site. Unlike other materials, prominent fibroblastproliferation and erythrocyte-filled blood vessels were found in the CTlayer. After 28 days, the healed TM remained thickened with atypicaldisorganized collagen fibres in the CT layer (FIG. 8).

The TM cross-sections were used to quantify changes in the TM thicknessfollowing treatment (FIG. 8). At 14 days, TMs in all groups weresubstantially thickened compared to normal TM (p<0.05) except SFStreated TM, which had similar thickness (14.13±4.04 μm) to the normal TM(p>0.05). By 28 days, statistically significant difference in TMthicknesses was found in the control, paper patch and GELFOAM® membranegroups (p<0.05). However, no statistically significant difference in TMthicknesses was seen between TYMPACOL™ membrane groups (8.55±4.25 μm)compared with the normal TM (p>0.05).

Transmission Electron Microscopy (TEM)

TEM was performed to investigate the microstructure of the healed TMs onday 28 post-repair. Briefly, following dissection, the perforation siteof the harvested TM samples was fixed in 2.5% glutaraldehyde and storedovernight at 4° C. Tissue specimens were washed, postfixed (1% osmicacid), dehydrated and embedded for transmission observation. Thintransverse sections were cut and examined with TEM (TECNAI 10, PhilipsCo., Netherlands) at 80 kV.

TEM observation was performed to investigate the ultrastructure ofhealed TMs 28 days post-surgery. In TYMPACOL™ membrane treated andspontaneously healed TMs, the CT layer was moderately thickened andfibroblast accumulation was apparent compared to the normal TM. InTYMPACOL™ membrane group, the three layers of the TM were readilyidentified, and the CT layer was compact with collagen bundleswell-orientated. However, in paper patch and GELFOAM® membrane groups,collagen fibres were loosely and irregularly arranged in the fibrouslayer, with obvious edema seen.

The medial aspect of TMs was observed with SEM to assess scaffoldattachment, cellular integration with scaffold and perforation closure.TYMPACOL™ membrane showed steady attachment to the perforation marginthroughout the healing process, thereby preserving their scaffoldfunction. TM epithelial cells migrated across the wound margin andadhered to the internal surface of to TYMPACOL™ membrane on day 5. By 9days, the TMs of TYMPACOL™ membrane group had healed and the internalsurface of neo-membranes was smooth. In contrast, paper patchdemonstrated early partial detachment from the TM surface, but itsscaffold function partially lost. Exudate formation and inflammatorycell infiltration was evident at the perforation site in the papergroup. GELFOAM® membrane showed early disintegration of its spongestructure. As shrinkage and absorption progressed, most of the GELFOAM®membrane dissolved, resulting in loss of its support function. Thehealed TMs in paper and GELFOAM® membrane groups showed some scarring at14 days. In the control group with no scaffold implantation, a rolledperforation edge of the unhealed TM was visible at 9 days. The TMeventually healed by 14 days, but with an obvious scar.

Auditory Brainstem Responses (ABR)

To assess the hearing of rats following grafting, ABR was performedusing the Nihon Kohden Neuropack-μ Measuring Systems (MEB-9100, NihonKoden, Japan) in a soundproof room. Rats were anesthetized beforetesting as previously described. Platinum subdermal needle electrodeswere inserted at the scalp vertex (active electrode), both mastoids(reference electrode) and at the nose tip (ground electrode). The teststimuli (click) with 0.1 ms duration were presented through an insertearphone. Animals were presented with a stimulus intensity series from90- to 0 dB sound pressure level (SPL) in 10 dB decrements. A total of512 responses were averaged in each series of stimuli over a 10 msanalysis period. Thresholds were defined as the lowest intensity toelicit a reproducible ABR waveform with typical wave III or wave IVmorphology. Auditory thresholds of click stimuli were measured pre- andpost TM perforation in the right ear of all rats, and at each time pointafter myringoplasty for the five animals from each group. The normalears and the ears with TM perforation without materials served ascontrols.

Hearing thresholds were similar in all treatment groups that measuredpre-perforation (p>0.05) as well as post-perforation (p>0.05). Theaverage auditory threshold of normal rat was 15.0 dB and this wassignificantly increased to 29.5 dB after perforation, indicating that TMperforation caused significant hearing loss (p<0.01). Audiometricassessment using ABR demonstrated hearing recovery for all groupsfollowing treatment (FIG. 9). The hearing recovery was defined as thedifference between auditory threshold immediately following perforation(pre-repair) and at specific time points following grafting(post-repair). Auditory threshold of all rats recovered over time, andsignificant differences were observed when comparing between differenttreatments (p<0.01). Most obviously, hearing in the animals treated withTYMPACOL™ membrane recovered significantly faster compared to thosetreated with paper patch (p<0.01), GELFOAM® membrane (p<0.01) andspontaneous healing (p<0.01).

Statistical Analysis

Healing rates determined by otoscopic observation were compared usingthe chi-square (χ²) test. Statistical analysis for ABR and TM thicknesswas evaluated using one-way analysis of variance (ANOVA) whereas hearingrecovery in each group over time was performed using multiple linearregression analysis. All analyses were performed using the StatisticalSoftware R (Version 2.11.1, package meta). Statistical significance wasdefined as p<0.05.

Conclusion

This study demonstrated that the collagen membrane of the presentinvention (TYMPACOL™ membrane) significantly shortened the perforationclosure time and promoted TM wound healing compared to two commonly usedscaffolds (paper patch and GELFOAM® membrane) and spontaneous healing ina rat model. The healed TMs in TYMPACOL™ membrane groups showed improvedmorphology with regeneration of compact collagen fibres, rapid return toa normal TM thickness, as well as complete hearing recovery at anearlier stage compared to the other groups. As the goals of surgicaltreatment for TM perforation are to achieve complete closure of theperforation and restoration of the hearing, these results suggest thatTYMPACOL™ membrane is efficient and will serve as an ideal scaffold torestore both TM healing and hearing.

Biocompatibility of a scaffold is an important element to consider, asinflammatory response following the application of biomaterials may leadto failure in surgery. Collagen is also known to elicit minimalinflammatory and antigenic responses. Pachence. (1996), J. biomed. Mat.Res.; 33:35-40). In this study, the TYMPACOL™ membrane accelerated andimproved TM healing, partly attributed to minimal inflammatory responseat the implantation sites.

In this study, we showed that TYMPACOL™ membrane achieved significantlyfaster hearing recovery compared to the other groups. We postulate thatthese improvement result from improved organization of collagen fibresof healed TMs and early remodelling to achieve comparable thickness to anormal TM.

TYMPACOL™ membrane was found to be easy to handle during surgery as itwas not as fragile as paper or bulky and spongy as GELFOAM® membrane.Moreover, the transparency of TYMPACOL™ membrane allowed directobservation of the TM, whereas the opacity of paper and GELFOAM®membrane obstructed the direct visibility of TM healing. From a clinicalpoint of view, these characteristics make TYMPACOL™ membrane morefavorable compared to paper and GELFOAM® membrane.

The invention claimed is:
 1. A method of producing a collagen-containingmembrane comprising the steps of: (i) isolating a collagen-containingtissue and incubating the isolated collagen-containing tissue in anethanol solution; (ii) incubating the isolated collagen-containingtissue from step (i) in a solution comprising about 1% (v/v) SDS andabout 0.2% (v/v) LiCl in order to denature non-collagenous proteinscontained therein; (iii) incubating the collagen-containing tissueproduced in step (ii) in a solution comprising about 0.5% (v/v) HCluntil the collagen in said material is denatured; and (iv) incubatingthe collagen-containing tissue produced in step (iii) in a solutioncomprising about 1% (v/v) HCl with simultaneous mechanical stimulationfor sufficient time to enable the collagen bundles in saidcollagen-containing tissue to align; wherein the mechanical stimulationcomprises applying tension cyclically to the collagen-containing tissue,thereby producing said collagen-containing membrane.
 2. The method ofclaim 1, wherein the ethanol solution comprises greater than 70%ethanol.
 3. The method of claim 1, wherein the incubation in step (ii)is at about 4° C.
 4. The method of claim 1, wherein the incubation instep (ii) is undertaken for at least 12 hours.
 5. The method of claim 1,wherein the incubation in step (iii) is undertaken for about 30 minutes.6. The method of claim 1, wherein the incubation in step (iii) isundertaken with shaking.
 7. The method of claim 1, wherein theincubation in step (iv) is undertaken for about 12 to 36 hours.
 8. Themethod of claim 7, wherein the incubation in step (iv) is undertaken forabout 24 hours.
 9. The method of claim 1, wherein the incubation in step(iv) is undertaken with shaking.
 10. The method of claim 1, furthercomprising a neutralization step between step (iii) and step (iv) whichcomprises incubation of said collagen-containing tissue with about 0.5%(v/v) NaOH.
 11. The method of claim 1, further comprising step (v) whichcomprises incubating the collagen-containing tissue from step (iv) withacetone and then drying the collagen-containing tissue.
 12. The methodof claim 1, wherein between steps (ii) and (iii) and/or between steps(iii) and (iv) the collagen-containing tissue is contacted with glycerolin order to visualise and facilitate the removal of fat and/or bloodvessels.
 13. The method of claim 12, wherein the glycerol is contactedwith the collagen-containing tissue for at least 10 minutes.
 14. Themethod of claim 1, wherein between steps (ii) and (iii) and/or betweensteps (iii) and (iv) the collagen-containing tissue is washed.
 15. Themethod of claim 14, wherein the wash used between steps (ii) and (iii)is acetone to remove the denatured proteins.
 16. The method of claim 15,wherein the collagen-containing tissue is further washed after theacetone with sterile water.
 17. The method of claim 15, wherein thecollagen-containing tissue is further washed in a NaOH:NaCl solution.18. The method of claim 1, wherein after steps (iv) thecollagen-containing tissue is washed with sterile water.
 19. The methodof claim 18, wherein the collagen-containing tissue is further washedafter the sterile water with the first solution.
 20. The method of claim1, wherein the collagen-containing tissue comprises dense connectivetissue.
 21. The method of claim 1, wherein the collagen-containingtissue is isolated from a sheep, a cow, a pig or a human.
 22. The methodof claim 1, wherein the collagen-containing tissue is isolated from ahuman.
 23. The method of claim 1, wherein the collagen-containing tissueis autologous.